| Metadata |
| datasetIdentifier | PASS00211 |
| datasetType | MSMS |
| submitter | Peter van Veelen <p.a.van_veelen@lumc.nl> |
| submitter_organization | |
| lab_head_full_name | |
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| datasetTag | Bcellligandome |
| datasetTitle | The Human Leukocyte Antigen-presented Ligandome of B lymphocytes |
| publicReleaseDate | 2013-04-01 00:00:00 |
| finalizedDate | 2013-03-06 05:46:20 |
| summary | Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies on T cell action, and in cellular immunotherapy and transplantation. In this study we present the in-depth identification and relative quantification of 15,500 peptide ligands constituting the HLA-ligandome of B-cells. This large number of identified ligands provides a general insight in the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA-ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes and post-translationally modified HLA ligands and can be a good starting point to solve a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes, not based on in silico predictions, but based on the bona fide eluted HLA-ligandome. |
| contributors | Chopie Hassan, Michel G.D. Kester, Arnoud H. de Ru, Pleun Hombrink, Jan Wouter Drijfhout, Harm Nijveen, Jack A.M. Leunissen, Mirjam, H.M. Heemskerk, J.H. Frederik Falkenburg, Peter A. van Veelen |
| publication | Chopie Hassan, Michel G.D. Kester, Arnoud H. de Ru, Pleun Hombrink, Jan Wouter Drijfhout, Harm Nijveen, Jack A.M. Leunissen, Mirjam, H.M. Heemskerk, J.H. Frederik Falkenburg, Peter A. van Veelen, Molecular Cellular Proteomics, accepted |
| growth | The Epstein-Barr virus (EBV) transformed B lymphoblastic cell lines B-LCL-HHC (typing: HLA-A*0201, B*0702, B*4402, Cw*0501 & Cw*0702) and B-LCL-JY pp65 (typing: HLA-A*0201, B*0702 & Cw*0702) were used as source of HLA-class I molecules. The CMV-derived pp65 transduced cell line was used to introduce an internal control for the ligandome since the CMVpp65-derived T cell epitopes are known (20). Cells were expanded in roller bottles using IMDM supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin and L-glutamine, were collected, washed with ice cold PBS, and stored at -80oC until use.
The hybridoma cell line was expanded in roller bottles to obtain W6/32 (anti HLA-Class I) antibody using protein free hybridoma medium supplemented with 1% penicillin/streptomycin, and 1.5 % L-glutamine. Antibodies produced by the hybridoma cell lines were purified from the supernatant using Prot-A sepharose beads, and eluted from the Prot-A beads with Glycine pH 2.5. The eluted antibodies were used to produce immunoaffinity column (W6/32- Prot-A sepharose 2.5 mg/ml). The W6/32 antibodies were covalently bound to Prot-A sepharose beads using dimethylpimelimidate (DMP). The columns were stored in PBS pH 8.0 and 0.02% NaN3 at 4 oC. |
| treatment | See above and below |
| extraction | Isolation of HLA class I-presented peptides-Extraction of peptides associated with HLA-class I molecules was performed as described (8,21). Briefly, pellets from 60 x109 B-LCL-JYpp65 cells and 40 x109 B-LCL-HHC cells were lysed in 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, and 0.5% Nonidet-P40, (pH 8.0) and supplemented with Complete® protease inhibitor (Sigma Aldrich). The total concentration of the cells in the lysis buffer was 0.1x 109 cells/ml.
After 2 hours incubation with tumbling of the cells in the lysis buffer at 4 oC, the preparation was centrifuged for 10 minutes at 2,500 rpm and 4 oC. The supernatant was transferred to a new tube and centrifuged for 35 minutes at 11,000 rpm and 4oC. The supernatant was pre-cleared with CL4B beads and subjected to the immunoaffinity column with a flow rate of 2.5 ml/min. After washing, bound HLA-class I/peptide complexes were eluted from the column, and dissociated with 10% acetic acid. Peptides were separated from the HLA-class I molecules by passage through a 10 kDa membrane (Pall macrosep centrifuge devices). The filtrate was freeze dried. If an oily sample remained after freeze drying, the sample was dissolved and the peptides were further purified by solid phase extraction (C18 Oasis, 100 µl bed volume, Waters). The peptides were eluted from the C18 Oasis column with 500 µl 50/50/0.1 water/ACN/FA, v/v/v. The eluted peptides from B-LCL-HHC cell lines were divided into two equal portions, freeze dried and dissolved in 95/3/0.1 water/ACN/FA, v/v/v. The eluted peptides from B-LCL-JYpp65 cell lines were divided into three equal portions, freeze dried, and dissolved in 95/3/0.1 water/ACN/FA, v/v/v. |
| separation | Peptide separation-For peptide IEF separations, the OFFGEL Agilent 3100 fractionator (Agilent Technologies, Waldbronn, Germany) was used. A modified method was applied by addition of 1 M urea to the buffer sample and rehydration buffer, instead of 5% glycerol only. The commercially available 13 cm IPG dry strips with a linear pH gradient ranging from 3-10 (GE-Health care) were used. The strips were rehydrated with 40 µL/well rehydration solution in the assembled device for 30 min. 150 µL of the prepared samples were loaded on each well, the cover fluid (mineral oil, Agilent technologies) was added onto both ends of the gel strip. The focusing methods, OG12PE01, as supplied by the manufacturer was applied for 12 well fractionations. The performance of the 3100 OFFGEL fractionator was checked under similar conditions in a separate run by determination of the pH using a pH indicator pH 3-10 (Fluka Analytical, Germany). Fractions were recovered and desalted by solid phase extraction, to desalt the fractions and to avoid the presence of oil and gel pieces in the samples, using C18 Oasis columns. The column was prewetted with 10/90 water/ACN v/v and equilibrated with 95/3/0.1 water/ACN/FA v/v/v. The samples were eluted with 50/50/0.1 water/ACN/FA v/v/v, freeze dried and dissolved in 100 µL 95/3/0.1 water/ACN/FA v/v/v. The twelve fractions generated from IEF were analyzed in triplicate and duplicate for B-LCL-HHC and B-LCL-JYpp65 cell lines, respectively, with nanoLC-MS/MS.
Next to the peptide IEF separation, two chromatographic separation techniques were applied, strong cation chromatography (SCX) and RP-C18 chromatography. For SCX separations, one portion of the eluted peptides from the W6/32 column was fractionated with a home-made SCX column (320 µm ID, 15 cm, polysulfoethyl A 3 um, Poly LC), run at 4 ul/min. Gradients were run for 10 min at 100% solvent A (100% water/0.1% TFA), after which a linear gradient started to reach 100% solvent B (250 mM KCl, 35% ACN/0.1% TFA) in 15 min, followed by 100 % solvent C (500 mM KCl, 35% ACN/0.1% TFA) in the next 15 min and remained at 100 % solvent C for 5 min, then switched again to 100 % solvent A. Twenty 4 ul-fractions were collected in vials prefilled with100 µl 95/3/0.1 water/ACN/FA v/v/v.
One portion of the eluted peptides from B-LCL-JY pp65 cell lines was fractionated on a home-made RP Reprosil-Pur C18-AQ column (200 µm ID, 3 µm x 15 cm) (Dr. Maisch, GmbH, Ammerbuch, Germany). The sample was loaded in solvent A (10/90/0.1 water/ACN/FA v/v/v), and the gradient was run from 0-50% B (10/90/0.1 water/ACN/FA v/v/v) in 30 min at a flow rate of 3 ul/min. The samples were taken up in a make-up flow of 50/50/0.1 water/ACN/FA at 100 ul/min supplied via a T-piece through the annular space between the separation capillary and an auxillary capillary. In this way 45 half a minute wide fractions were collected, subsequently freeze dried, and dissolved in solvent A for analysis by nanoLC-MS/MS. |
| digestion | not applicable. Ligandomics study |
| acquisition | LC-MS/MS analysis-The dissolved fractions were analyzed by on-line nano-HPLC mass spectrometry with a system, consisting of a conventional Agilent 1100 gradient HPLC system (Agilent, Waldbronn, Germany), described by Meiring et al (22), and a LTQ-FT Ultra mass spectrometer (Thermo, Bremen, Germany). Fractions were injected onto a home-made precolumn (100 um×15 mm; Reprosil-Pur C18-AQ 3 µ m, Dr Maisch, Ammerbuch, Germany) and eluted via in home-made analytical nano-HPLC column (15 cm×50 µm; Reprosil-Pur C18-AQ 3 um). The gradient was run from 0% to 50% solvent B (10/90/0.1 water/ACN/FA v/v/v) in 90 min. The nano-HPLC column was drawn to a tip of approximately 5 µm and acted as the electrospray needle of the MS source. The mass spectrometer was operated in data dependent mode, automatically switching between MS and MS/MS acquisition. Full scan mass spectra were acquired in the FT-ICR with a resolution of 25,000 at a target value of 5,000,000. The two most intense ions were then isolated for accurate mass measurements by a selected ion monitoring scan in FT-ICR with a resolution of 50,000 at a target accumulation value of 50,000. The selected ions were then fragmented in the linear ion trap using collision-induced dissociation at a target value of 10,000. In a post analysis process, raw data were converted to peak lists using Bioworks Browser software, Version 3.2.0. |
| informatics | Data analysis-The tandem mass spectra were matched against the International Protein Index (IPI) human database version 3.87, using the mascot search engine version 2.2.04 (Matrix Science, London, UK)), with a precursor mass tolerance of 2 ppm, with methione oxidation as a variable modification, and a product ion tolerance of 0.8 Da. For finding post-translationally modified HLA-ligands phosphorylation on serine, threonine and tyrosine were allowed, and cysteinylation of cysteine in separate searches. Scaffold software version 3 (www.proteomesoftware.com) was subsequently used to process the mascot output files and generate spectrum reports. Duplicates were removed, and peptides with a best mascot ion score >35 and 8-11 amino acids long, were selected for the production of supplemental Table S1. For the immunological examples a best mascot score of >20 was selected, and the length was restricted to 8-18 amino acids. Next to the above mentioned procedure, Proteome Discoverer 1.3 (Thermo, Bremen, Germany) was used to extract all identified peptides from the input *.RAW-files, using the mascot server mentioned above, and calculate their intensity, as reported in supplemental Table S1. False discovery rates were as determined by Proteome Discoverer, for the Homo Sapiens-extracted Uniprot/SWISSProt database (23) (release 2010_11) (www.ebi.ac.uk) containing 20,259 protein sequences, and the IPIhuman3.87 database (www.ebi.ac.uk/IPI/IPIhuman.html) see also supplemental Table S2. Icelogo version 1.2 was used to generate the binding motifs as presented in Fig. 5 and supplemental Fig. S1 (24). The GRAVY index was calculated using http://www.bioinformatics.org/sms2/ protein_gravy.html. The pIs of the identified peptides were calculated using http://www.expasy.org/tools/pi_tool.html. NetMHC 3.2 (http://www.cbs.dtu.dk/services/NetMHC) was used to predict the binding affinity (nM) of the identified peptides to HLA-A*0201, HLA-B*0702 and HLA-B*4402. Overall protein turnover values were taken from Cambridge et al. (25). Phosphosite.org (26)(www.phosphosite.org) and Phospho.ELM 8.3 (27) (http://phospho.elm.eu.org) were used to find known phosphosites in the identified peptides. NetPhos 2.0 (28) (http://www.cbs.dtu.dk/services/NetPhos/) was used to predict phosphosites in identified phosphorylated ligands. For the identification of polymorphic peptides, the tandem mass spectra were matched against the HSPVdb, a database optimized for finding polymorphic peptides (16). For searching CMV pp65-derived ligands/epitopes in B-LCL-JYpp65, a separate database was constructed only containing the DNA sequence of the pp65 protein (NCBI; pp65_AD169_seq with intron Human herpesvirus 5, complete genome). For searching EBV-derived epitopes, a separate database was constructed containing the DNA sequence of EBV selected from RefSeq database (29) (>gi|82503188|ref|NC_007605.1| Human herpesvirus 4 type 1, complete genome). |
| instruments | LTQ-FT Ultra |
| species | Human |
| massModifications | variable: M+15.994915,M+79.966331, M+119,1423 |
